The invention relates to L-lysine-producing strains of corynebacteria with enhanced lysE gene (lysine export carrier gene), in which strains additional genes, chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapB gene (dihydrodipicolinate reductase gene) and the pyc gene, but especially the dapA gene and the lysC gene (aspartate kinase gene), are amplified and, in particular, over-expressed, and to a process for the preparation of L-lysine.
L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition. The need has been steadily increasing in recent years.
L-Lysine is prepared by a fermentation process with L-lysine-producing strains of corynebacteria, especially Corynebacterium glutamicum. Because of the great importance of this product, attempts are constantly being made to improve the preparative process. Improvements to the process may relate to measures involving the fermentation technology, e.g. stirring and oxygen supply, or the composition of the nutrient media, e.g. the sugar concentration during fermentation, or the work-up to the product form, e.g. by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself.
The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. S-(2-aminoethyl)cysteine, or auxotrophic for amino acids, e.g. L-leucine, and produce L-lysine.
Methods of recombinant DNA technology have also been used for some years in order to improve L-lysine-producing strains of Corynebacterium glutamicum by amplifying individual biosynthesis genes and studying the effect on L-lysine production.
Thus, EP-A-0 088 166 reports the increase in productivity, after amplification, of a DNA fragment conferring resistance to aminoethylcysteine. EP-B-0 387 527 reports the increase in productivity, after amplification, of an lysC allele coding for a feedback-resistant aspartate kinase. EP-B-0 197 335 reports the increase in productivity, after amplification, of the dapA gene coding for dihydrodipicolinate synthase. EP-A-0 219 027 reports the increase in productivity, after amplification, of the asd gene coding for aspartate semialdehyde dehydrogenase. Pisabarro et al. (Journal of Bacteriology 175(9), 2743-2749 (1993)) describe the dapB gene coding for dihydrodipicolinate reductase.
The effect of the amplification of primary metabolism genes on L-lysine production has also been studied. Thus EP-A-0 219 027 reports the increase in productivity, after amplification, of the aspC gene coding for aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940 report the increase in productivity, after amplification, of the ppc gene coding for phosphoenolpyruvate carboxylase. DE-A-198 31 609 reports the increase in productivity, after amplification, of the pyc gene coding for pyruvate carboxylase.
Finally, DE-A-195 48 222 describes that an increased activity of the L-lysine export carrier coded for by the lysE gene promotes lysine production.
In addition to these attempts to amplify an individual gene, attempts have also been made to amplify two or more genes simultaneously and thereby to improve L-lysine production in corynebacteria. Thus, DE-A-38 23 451 reports the increase in productivity, after simultaneous amplification, of the asd gene and the dapA gene from Escherichia coli. DE-A-39 43 117 discloses the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapA gene by means of plasmid pJC50. EP-A-0 841 395 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapB gene; further improvements could be achieved by additional amplification of the dapB, lysA and ddh genes. EP-A-0 854 189 describes the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase and of the dapA, dapB, lysA and aspC genes. EP-A-0 857 784 particularly reports the increase in productivity, after simultaneous amplification, of an lysC allele coding for a feedback-resistant aspartate kinase enzym and of the lysA gene; a further improvement could be achieved by additional amplification of the ppc gene.
It is clear from the many processes described in the state of the art that there is a need for the development of novel approaches and for the improvement of existing processes for lysine production with corynebacteria.
The object of the invention consists in using novel measures to provide improved L-lysine-producing strains of corynebacteria.
L-Lysine is a commercially important L-amino acid which is used especially as a feed additive in animal nutrition.
When L-lysine or lysine is mentioned in the following text, it is understood as meaning not only the base but also the appropriate salts, e.g. lysine hydrochloride or lysine sulfate.
The invention provides L-lysine-producing strains of corynebacteria enhanced lysE gene (lysine export carrier gene), wherein they additionally contain genes chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapB gene (dihydrodipicolinate reductase gene) and the pyc gene (pyruvate carboxylase gene), but especially the dapA gene and the lysC gene, which, individually or together, are enhanced and, preferably, over-expressed.
The novel DNA sequence located upstream (5xe2x80x2 end) from the dapB gene has also been found which carries the xe2x88x9235 region of the dapB promoter and is advantageous for the expression of the dapB gene. It is shown as SEQ ID No. 1.
A corresponding DNA capable of replication, with the nucleotide sequence shown in SEQ ID No. 1, is therefore claimed as well.
The invention also provides the MC20 or MA16 mutations of the dapA promoter shown in SEQ ID No. 5 and SEQ ID No. 6, deposited under DSM12868 and DSM12867 respectively.
The invention also provides L-lysine-producing strains of corynebacteria with enhanced lysE gene, wherein additionally the dapA and dapB genes are simultaneously enhanced and, in particular, over-expressed.
Finally, the invention also provides L-lysine-producing strains of corynebacteria with enhanced lysE gene, wherein additionally the dapA and lysC genes are simultaneously enhanced and, in particular, over-expressed.
In this context the term xe2x80x9cenhancementxe2x80x9d describes the increase in the intracellular activity, in a microorganism, of one or more enzymes which are coded for by the appropriate DNA, by increasing the copy number of the gene(s), using a strong promoter or using a gene coding for an appropriate enzyme with a high activity, and optionally combining these measures.
In this context, xe2x80x9camplificationxe2x80x9d describes a specific procedure for achieving an enhancement whereby the number of DNA molecules carrying a gene or genes, an allele or alleles, a regulatory signal or signals or any other genetic feature(s) is increased.
A process for the preparation of L-lysine by the fermentation of these corynebacteria is also claimed.
The microorganisms which the present invention provides can prepare L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch or cellulose or from glycerol and ethanol, especially from glucose or sucrose. Said microorganisms are corynebacteria, especially of the genus Corynebacterium. The species Corynebacterium glutamicum may be mentioned in particular in the genus Corynebacterium, being known to those skilled in the art for its ability to produce amino acids. This species includes wild-type strains such as Corynebacterium glutamicum ATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecola ATCC17965 and strains or mutants derived therefrom. Examples of L-lysine-producing mutants of corynebacteria are:
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Brevibacterium flavum FERM-P 6463
Brevibacterium flavum FERM-P 6464
Corynebacterium glutamicum DSM5714
Corynebacterium glutamicum DSM12866
DE-A-195 48 222 discloses the advantageous effect of over-expression of the lysE gene on L-lysine production.
The additional enhanced expression of the dapB gene or the pyc gene, or in particular an additionally enhanced expression of an lysC allele coding for a feedback-resistant aspartate kinase, or in particular an additionally enhanced expression of the dapA gene, improves L-lysine production.
The inventors have also found that, for a given over-expression of the lysE gene, the simultaneous, additionally enhanced expression of the dapA and dapB genes brings further advantages for L-lysine production.
A corresponding DNA capable of replication, with the nucleotide sequence shown in SEQ ID No. 1, is therefore claimed as well.
For a given over-expression of the lysE gene, the simultaneous, additionally enhanced expression of the dapA gene and the lysC allele is also advantageous.
An enhancement (over-expression) is achieved e.g. by increasing the copy number of the appropriate genes or mutating the promoter and regulatory region or the ribosome binding site located upstream from the structural gene. Expression cassettes incorporated upstream from the structural gene work in the same way. Inducible promoters additionally make it possible to increase the expression in the course of the formation of L-lysine by fermentation. Measures for prolonging the life of the mRNA also improve the expression. Furthermore, the enzyme activity is also enhanced by preventing the degradation of the enzyme protein, the genes or gene constructs either being located in plasmids (shuttle vectors) of variable copy number or being integrated and amplified in the chromosome. Alternatively, it is also possible to achieve an over-expression of the genes in question by changing the composition of the media and the culture technique.
Those skilled in the art will find relevant instructions inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), EP-0 472 869, U.S. Pat. No. 4,601,893, Schwarzer and Pxc3xchler (Bio/Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)) or the handbook xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981) and well-known textbooks on genetics and molecular biology.
The genes from Corynebacterium glutamicum used according to the invention are described and can be isolated, prepared or synthesized by known methods.
Methods of localized mutagenesis are described inter alia by Higuchi et al. (Nucleic Acids Research 16: 7351-7367 (1988)) or by Silver et al. in the handbook by Innis, Glefand and Sninsky (eds.) entitled PCR Strategies (Academic Press, London, UK, 1995).
The first step in isolating a gene of interest from C. glutamicum is to construct a gene library of this microorganism in e.g. E. coli or optionally also in C. glutamicum. The construction of gene libraries is documented in generally well-known textbooks and handbooks. Examples which may be mentioned are the textbook by Winnacker entitled From Genes to Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al. entitled Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). Bathe et al. (Molecular and General Genetics 252: 255-265 (1996)) describe a gene library of C. glutamicum ATCC13032 which was constructed using cosmid vector SuperCos I (Wahl et al., Proceedings of the National Academy of Sciences USA, 84: 2160-2164 (1987)) in E. coli K-12 NM554 (Raleigh et al., Nucleic Acids Research 16: 1563-1575 (1988)). Bxc3x6rmann et al. (Molecular Microbiology 6(3), 317-326) in turn describe a gene library of C. glutamicum ATCC13032 using cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).
A gene library of C. glutamicum in E. coli can also be constructed using plasmids like pBR322 (Bolivar, Life Sciences 25, 807-818 (1979)) or pUC19 (Norrander et al., Gene, 26: 101-106 (1983)). In the same way it is also possible to use shuttle vectors such as pJC1 (Cremer et al., Molecular and General Genetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., Gene 102, 93-98 (1991)), which replicate in E. coli and C. glutamicum. Restriction- and/or recombination-defective strains are particularly suitable hosts, an example being the E. coli strain DH5xcex1mcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences, USA 87, 4645-4649 (1990)). Other examples are the restriction-defective C. glutamicum strains RM3 and RM4, which are described by Schxc3xa4fer et al. (Applied and Environmental Microbiology 60(2), 756-759 (1994)).
The gene library is then transferred to an indicator strain by transformation (Hanahan, Journal of Molecular Biology 166, 557-580 (1983)) or electroporation (Tauch et al., FEMS Microbiological Letters, 123: 343-347 (1994)). The characteristic feature of the indicator strain is that it possesses a mutation in the gene of interest which causes a detectable phenotype, e.g. an auxotrophy. The indicator strains or mutants are obtainable from publicized sources or strain collections, e.g. the Genetic Stock Center of Yale University (New Haven, Conn., USA), or if necessary are specially prepared. An example of such an indicator strain which may be mentioned is the E. coli strain RDA8 requiring mesodiaminopimelic acid (Richaud et al., C. R. Acad. Sci. Paris Ser. III 293: 507-512 (1981)), which carries a mutation (dapA::Mu) in the dapA gene.
After transformation of the indicator strain with a recombinant plasmid carrying the gene of interest, and expression of the gene in question, the indicator strain becomes prototrophic in respect of the appropriate characteristic. If the cloned DNA fragment confers resistance, e.g. to an antimetabolite like S-(2-aminoethyl)cysteine, the indicator strain carrying the recombinant plasmid can be identified by selection on appropriately supplemented nutrient media.
If the nucleotide sequence of the gene region of interest is known or obtainable from a data bank, the chromosomal DNA can be isolated by known methods, e.g. as described by Eikmanns et al. (Microbiology 140, 1817-1828 (1994)), and the gene in question can be synthesized by the polymerase chain reaction (PCR) using suitable primers and cloned into a suitable plasmid vector, e.g. pCRIITOPO from Invitrogen (Groningen, The Netherlands). A summary of PCR methodology can be found in the book by Newton and Graham entitled PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).
Examples of publicly accessible data banks for nucleotide sequences are that of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or that of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA).
The isolation and cloning of the lysE gene from C. glutamicum ATCC13032, together with the nucleotide sequence, are described in DE-A-195 48 222.
The isolation, cloning and sequencing of the dapA gene from various strains of C. glutamicum are described by Cremer et al. (Molecular and General Genetics 220: 478-480 (1990)), by Pisabarro et al. (Journal of Bacteriology 175: 2743-2749 (1993)) and by Bonnassie et al. (Nucleic Acids Research 18, 6421 (1990)). The nucleotide sequence of the dapA gene is obtainable under accession number X53993.
The isolation, cloning and sequencing of the dapB gene from Brevibacterium lactofermentum are described by Pisabarro et al. (Journal of Bacteriology 175: 2743-2749 (1993)). The nucleotide sequence of the dapB gene is obtainable under accession number X67737.
The isolation, cloning and sequencing of the lysC gene and of lysC alleles coding for a feedback-resistant aspartate kinase are reported by several authors. Thus, Kalinowski et al. (Molecular and General Genetics 224: 317-324 (1990)) report the lysC allele from the C. glutamicum strain DM58-1. DE-A-39 43 117 reports the cloning of the lysC allele from the C. glutamicum strain MH20. Follettie et al. (Journal of Bacteriology 175: 4096-4103 (1993)) report the lysC allele from the C. flavum strain N13, which is called ask in said publication. Kalinowski et al. (Molecular Microbiology 5, 1197-1204 (1991)) report the lysC gene from C. glutamicum ATCC13032. The nucleotide sequences of the lysC gene and of various lysC alleles are obtainable inter alia under accession numbers X57226 and E06826.
The genes obtained in this way can then be incorporated inter alia into plasmid vectors, e.g. pJC1 (Cremer et al., Molecular and General Genetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., Gene 102, 93-98 (1991)), individually or in suitable combinations, transferred to desired strains of corynebacteria, e.g. the strain MH20-22B (Schrumpf et al., Applied Microbiology and Biotechnology 37: 566-571 (1992)), by transformation, e.g. as in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), or by electroporation, e.g. as in Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)), and expressed. The strain to be chosen can equally well be transformed with two plasmid vectors, each containing the gene or genes in question, thereby achieving the advantageous, simultaneously enhanced expression of two or more genes in addition to the known enhancement of the lysE gene.
Examples of such strains are:
the strain MH20-22B/pJC33/pEC7lysE, in which the lysE and lysC genes are expressed with simultaneous enhancement, or
the strain MH20-22B/pJC50/pEC7lysE, in which the lysE, lysC and dapA genes are expressed with simultaneous enhancement, or
the strain MH20-22B/pJC23/pEC7lysE, in which the lysE and dapA genes are expressed with simultaneous enhancement, or
the strain MH20-22B/pJC23/pEC7dapBlysE, in which the lysE, dapA and dapB genes are expressed with simultaneous enhancement.
The microorganisms prepared according to the invention can be cultivated for L-lysine production continuously or discontinuously by the batch process, the fed batch process or the repeated fed batch process. A summary of known cultivation methods is provided in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Bioprocess Technology 1. Introduction to Bioengineering) (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Bioreactors and Peripheral Equipment) (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).
The culture medium to be used must appropriately meet the demands of the particular microorganisms. Descriptions of culture media for various microorganisms can be found in the handbook xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981).
Carbon sources which can be used are sugars and carbohydrates, e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, e.g. palmitic acid, stearic acid and linoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids, e.g. acetic acid. These substances can be used individually or as a mixture.
Nitrogen sources which can be used are organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.
Phosphorus sources which can be used are potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium salts. The culture medium must also contain metal salts, e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins can be used in addition to the substances mentioned above. Said feed materials can be added to the culture all at once or fed in appropriately during cultivation.
The pH of the culture is controlled by the appropriate use of basic compounds such as sodium hydroxide, potassium hydroxide or ammonia, or acid compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled using antifoams such as fatty acid polyglycol esters. The stability of plasmids can be maintained by optionally adding suitable selectively acting substances, e.g. antibiotics, to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gaseous mixtures, e.g. air, into the culture. The temperature of the culture is normally 20xc2x0 C. to 45xc2x0 C. and preferably 25xc2x0 C. to 40xc2x0 C. The culture is continued until L-lysine formation has reached a maximum. This objective is normally achieved within 10 hours to 160 hours.
The concentration of L-lysine formed can be determined with the aid of amino acid analyzers by means of ion exchange chromatography and postcolumn reaction with ninhydrin detection, as described by Spackmann et al. (Analytical Chemistry 30, 1190 (1958)).
The following microorganisms have been deposited in the Deutsche Sammlung fxc3xcr Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures (DSMZ), Brunswick, Germany) under the terms of the Budapest Treaty:
Escherichia coli K-12 strain DH5xcex1/pEC7lysE as DSM12871
Escherichia coli K-12 strain DH5xcex1/pEC7dapBlysE as DSM12875
Corynebacterium glutamicum strain DSM5715/pJC23 as DSM12869
Corynebacterium glutamicum strain DSM5715aecD::dapA(MA16) as DSM12867
Corynebacterium glutamicum strain DSM5715aecD::dapA(MC20) as DSM12868